Single-molecule detection of biomolecules is a potentially useful technology for diagnostic and biosensing applications. However, single-molecule observation in specimens that contain high concentrations of coexisting molecules poses technological challenges due to large background noise. We developed a nanometric gap structure combined with a fluid lipid membrane (nanogap-junction) for selectively detecting specific biological molecules in a solution having high concentrations of coexisting molecules. The nanogap-junction comprised a patterned model membrane (polymeric and fluid bilayers), an adhesion layer, and a polydimethylsiloxan (PDMS). By attaching the polymeric bilayer and the PDMS sheet with an adhesion layer having a finite thickness (lipid-coated silica nanoparticles), a nanometric gap was generated between the fluid bilayer and PDMS. In the present study, we developed a methodology to observe single molecules of non-labeled biomarker in the nanogap-junction. Prostate specific antigen (PSA), a prominent biomarker of the prostate cancer, was used as a model. We observed PSA molecules by the sandwich immunoassay method, using membrane-bound anti-PSA antibody (PS1) and fluorescently labeled anti-PSA antibody (PS2). PS1 was attached onto the fluid bilayer either by conjugating Fab’ with phospholipid via the click chemistry or by reconstituting pre-conjugated Fab’-lipid into the bilayer. Single molecules of PSA were observed to diffuse laterally on the lipid bilayer in the nanogap-junction. We could detect PSA at concentrations where observation by the total internal reflection fluorescence was not possible due to the high background noise. These results demonstrate the feasibility of nanogap-junction for highly sensitive detection. The nanogap-junction offers a versatile platform for broad biomedical applications by realizing highly selective and sensitive detection in samples containing diverse coexisting molecules.

Single molecule detection of biomarker in a nanometric gap structure combined with fluid membrane

255th National Meeting (2018)

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